The present invention relates to a method of reproducing plants by somatic embryogenesis involving placing embryogenic tissue onto a semi-permeable membrane which is on a culture medium and incubating to produce a normal somatic embryo.
Somatic embryogenesis has been widely demonstrated across the plant kingdom and includes angiosperm and gymnosperm, dicot and monocot, and herbaceous and woody plant species (Jain, S. M.; Gupta, P. K.; Newton, R. J. eds., Somatic embryogenesis in woody plants, vol. 1-history, molecular and biochemical aspects, and applications, Kluwer Academic Publishers, 1995; Thorpe, T. A., In vitro embryogenesis in plants, Kluwer Academic Publishers, 1995). The efficient production of somatic embryos is of considerable practical and biological interest compared to plants regenerated via organogenesis. Some of the prospective advantages of somatic embryogenesis include the following (Ammirato, P. V., Embryogenesis, In: Evans, E. A., Sharp, W. R., Ammirato, P. V., Yamada, Y., eds., Handbook of plant cell culture, Volume 1, Techniques for propagation and breeding, Macmillan Publishing Company, 1983:82-123): plant regeneration is more efficient since somatic embryos are bipolar structures and germination is a one-step process whereas organogenesis generally requires separate initiation of a shoot and root meristem; many more plants can be produced than is possible by organogenesis; somatic embryos can be encapsulated and treated like normal seed (i.e., stored and shipped); somatic embryos could be used for long-term storage in germplasm banks because of inherent dormancy properties; somatic embryos produce secondary metabolites not produced by undifferentiated callus (Niedz, R. P., et al., Plant Cell Tiss. Org. Cult., 51:181-185 (1997)); and somatic embryogenesis is a biological phenomenon uniquely suited as a tool to study basic questions of plant growth and development.
Citrus was one of the earliest plant genera where somatic embryogenesis was reported (Ranga Swamy, N. S., Experientia, 14:111-112 (1958); Maheshwari, P., and N. S. Ranga Swamy, Indian J. Hort., 15:275-282 (1958)). However, the routine production of large numbers of normal somatic embryos in alfalfa (McKersie, B. D., and Bowley, S. R., Synthetic seeds of alfalfa, In: Redenbaugh, K., eds., Synseeds: applications of synthetic seeds to crop improvement, CRC Press, 1993:231-256), carrot (Molle, F., et al., Carrot somatic embryogenesis and its application to synthetic seeds, In: Redenbaugh, K., eds., Synseeds: applications of synthetic seeds to crop improvement, CRC Press; 1993:257-287), or celery and lettuce (Sanada, M., et al., Celery and lettuce, In: Redenbaugh, K., eds. Synseeds: applications of synthetic seeds to crop improvement, CRC Press, 1993:305-327) is not possible for most species, including Citrus, where somatic embryogenesis has been reported. Globular stage embryos are readily produced from citrus embryogenic callus in large numbers. However, normalized development of these embryos has not been reported.
In accordance with the present invention there is provided a method of reproducing plants by somatic embryogenesis involving placing embryogenic tissue onto a semi-permeable membrane which is on a culture medium and incubating to produce a normal somatic embryo.